Compositions for inhibiting thrombin-induced coagulation and methods of use

ABSTRACT

A method of achieving safe and effective treatment or prevention of potentially harmful blood clots, or in inhibiting the coagulation of blood when so desired such as during a wide array of disease conditions including stroke, myocardial infarction, sickle-cell crisis and venous thrombosis, is provided by the administration of a fibrinogen-binding protein capable of binding at the N-terminal Bβ chain of fibrinogen, such as SdrG or Fbe, or their respective binding regions such as the A domain. In addition, compositions comprising effective amounts of the fibrinogen-binding proteins are also provided. The present anti-coagulation compositions have been shown to inhibit thrombin-induced fibrin clot formation by interfering with the release of fibrinopeptide B and the resulting anti-coagulation effects can be achieved without potential for causing or exacerbating unwanted side effects such as thrombocytopenia associated with prior art anticoagulants such as heparin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/142,935, filed May 13, 2002, which claims the benefit of U.S.Provisional Patent Application No. 60/290,072, filed May 11, 2001.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The subject matter of this application was supported by Grant No.AI20624 from the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates in general to SdrG, a fibrinogen-bindingbacterial adhesin, and in particular to the use of SdrG or its bindingregion as an anti-coagulation agent by virtue of its ability to inhibitthrombin-induced fibrin clot formation by interfering with the releaseof fibrinopeptide B. In addition, the invention relates to compositionsand methods utilizing SdrG or its binding region for inhibitingthrombin-induced coagulation and conditions associated therewith.

BACKGROUND OF THE INVENTION

Coagulase-negative staphylococci (CNS) are important opportunisticpathogens that are particularly associated with foreign body infectionsin humans. Staphylococcus epidermidis is the most common pathogenicspecies of CNS and accounts for 74-92% of the infections caused by thisgroup of staphylococci (1). The molecular pathogenesis of mostinfections is complex and involves multiple microbial factors and hostcomponents, but is generally initiated by the adherence of the microbeto host tissues. Bacterial adherence involves specific surfacecomponents called adhesins, and bacterial pathogens, such asstaphylococci that live in the extracellular space of the host, targetextracellular matrix (ECM) components, including fibrinogen (Fg) andfibronectin, for adherence and colonization. This process is mediated bya sub-family of adhesins that have been termed MSCRAMM®s (microbialsurface components recognizing adhesive matrix molecules) (2).Staphylococcus aureus expresses multiple MSCRAMM®s of which several havebeen characterized in some detail (For a recent review see Ref. 3), andvarious MSCRAMM®s have been the subject of U.S. Patents, includingfibronectin binding proteins such as disclosed in U.S. Pat. Nos.5,175,096; 5,320,951; 5,416,021; 5,440,014; 5,571,514; 5,652,217;5,707,702; 5,789,549; 5,840,846; 5,980,908; and 6,086,895; fibrinogenbinding proteins such as disclosed in U.S. Pat. Nos. 6,008,341 and6,177,084; and collagen binding proteins as disclosed in 5,851,794 and6,288,214; all of these patents incorporated herein by reference. Inaddition, other information concerning SdrG and other MSCRAMM®s can befound in U.S. Ser. No. 09/810,428, filed Mar. 19, 2001, incorporatedherein by reference; and U.S. Ser. No. 09/386,962, filed Aug. 31, 1999,incorporated herein by reference.

In addition to S. epidermidis, S. aureus also causes serious foreignbody infections. S. aureus appears to adhere to the biomaterial throughan indirect mechanism. Upon implantation, the foreign body rapidlybecomes coated with host proteins derived primarily from plasma with Fgbeing a dominant component. S. aureus appears to adhere to the absorbedproteins rather than to the biomaterial itself using adhesins of theMSCRAMM® family (4,5). At least four of the S. aureus MSCRAMM®srecognize Fg. Two of these MSCRAMM®s, clumping factor A and B (ClfA,ClfB), have Fg-binding A-regions followed by a long segment of Ser-Asp(SD) dipeptide repeats. The other two Fg-binding MSCRAMM®s, contain asimilar ligand binding A-region followed by a fibronectin binding motifthat is repeated 5 times (6). Because the fibronectin binding activitywas identified first, these two MSCRAMM®s are known as fibronectinbinding protein A and B (FnbpA and FnbpB) (7,8). Studies havedemonstrated the importance of ClfA and ClfB in the adherence of S.aureus to plasma-coated biomaterials. S. aureus mutants deficient in oneor both of these MSCRAMM®s exhibited an impaired ability to adhere toplasma-coated catheters in vivo or ex vivo (9,10).

For S. epidermidis, adherence to foreign bodies appears to involve bothspecific and non-specific processes. The bacteria may initiallyassociate directly with the foreign body through non-specificinteractions, while the later stages of adherence may involve morespecific interactions between bacterial adhesins and host ligands. S.epidermidis expresses polysaccharide adhesins including PS/A and PIA,which are encoded by the ica locus (11,12). In addition, the presentinventors (13) and others (14) have recently shown that S. epidermidiscontains surface proteins structurally related to S. aureus MSCRAMM®s.Two of these S. epidermidis proteins, called SdrF and SdrG, havefeatures typical of Gram-positive bacterial proteins that are anchoredto the cell wall. Both proteins show significant amino acid sequencehomology to ClfA and ClfB from S. aureus including an ˜500 amino acidlong A region, a SD dipeptide repeat region and features required forcell wall anchoring, including a LPXTG (SEQ ID NO:1) motif (FIG. 1A).Recent studies by Pei, et al. suggest that another S. epidermidisprotein, called Fbe, can bind Fg and, much like SdrG, specificallyrecognizes the Bβ chain of this molecule (15). However, this referencedoes not disclose or suggest the specific binding site for the Fbeprotein on fibrinogen and thus does not disclose or suggest that thebinding site for this protein would be related to of affect in anymanner the binding site for thrombin on fibrinogen.

Of these proteins from S. epidermidis, SdrG is of particular interestfor its ability to bind Fg. Fg is known to play a critical role in theformation of blood clots, although previously the precise binding siteof SdrG to Fg has not been localized with specificity. Accordingly,because the precise binding site for SdrG in the Fg Bβ chain has notbeen localized, it has not been previously been associated with thethrombin cleavage site on fibrinogen and thus it has not previously beenrecognized or suggested that SdrG might be useful in inhibiting thethrombin-induced cleavage of fibrinogen and the thrombin-induced processof clot formation.

In general, the blood clots generated by Fg, e.g. through its cleavageby thrombin to form fibrin and start the process of blood coagulation,are beneficial in the normal wound healing process. However, abnormalclots caused by the cleavage of Fg can lead to thrombosis, a conditionwhere a clot develops in the circulatory system. Thrombosis is anextremely dangerous condition and may produce ischemic necrosis of thetissue supplied by the artery, e.g., myocardial infarction due tothrombosis of a coronary artery, or stroke due to thrombosis of acerebral artery. In addition to the above, venous thrombosis may causethe tissues drained by the vein to become edematous and inflamed, andthrombosis of a deep vein may result in a pulmonary embolism. Stillother problems result in sickle-cell patients wherein the malformed“sickle cells” can also lead to a sickle-cell crisis state in whichcoagulation reaches dangerous proportions, and this can once againresult in serious injury or even death.

Generally, anticoagulant agents such as heparin and its derivatives areused to treat thrombosis and to prevent or reduce coagulation whendesirable such as in the case of myocardial infarction and the otherconditions discussed above. Heparin works by inhibiting thrombingeneration and in antagonizing thrombin's action. However, the use ofheparin has distinct problems which have yet to be overcome. Onedisadvantage associated with heparin is that it can only be administeredparenterally. Another serious disadvantage is major bleeding occurs in1% to 33% of patients who receive various forms of heparin therapy. Infact, purpura, ecchymoses, hematomas, gastrointestinal hemorrhage,hematuria, and retroperitoneal bleeding are regularly encounteredcomplications of heparin therapy. In addition to the abovecomplications, thrombocytopenia occurs in 1% to 5% of patients receivingheparin.

Accordingly, there is thus a distinct and growing need to providealternatives to heparin as anti-coagulation agent which do not sufferfrom all of the above-mentioned side effects or disadvantages. One suchalternative is the use of snake venom products including ancrod, anα-fibrinogenase isolated from Calloselasma rhodostoma (Malayan Pitviper). However, ancrod appears to release only FpA and leads to theformation of an unstable fibrin clot (Bell 1997). Moreover, because thisdefibrinating enzyme cleaves FpA and not FpB from Fg, it forms a clotthat is very sensitive to endogenous fibrinolysis, and additionallyactivates plasminogen further contributing to fibrinolysis (Pizzo,Schwartz et al. 1972; Carr 1975; Bell 1997). Hypofibrinogenemia, i.e.,the reduction of Fg in the blood, must be sustained by administeringancrod daily since after termination of treatment, the plasma Fg risesand returns to normal levels in days (Bell, Bolton et al. 1968). Thelimited clinical experience indicates that which defibrination may beachieved with ancrod, the elaboration of neutralizing antibodies withrepeated injections of ancrod leads to resistance (see, e.g., Pitney,Holt et al. 1969; Pitney and Regoeczi 1970), (Vinazzer 1973; Sapru, Mozaet al. 1975).

In short, there is a distinct and acute need to provide a safe andeffective alternative to the use of heparin in achieving the inhibitionin blood coagulation in cases wherein such inhibition may be crucial insaving the life of a human or animal patient.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide safe andeffective alternatives to the use of heparin in achieving therapeuticanti-coagulant treatment in human and animal patients.

It is further an object of the present invention to provide methods ofutilizing fibrinogen-binding proteins from S. epidermidis in theprevention or treatment of thrombin-induced coagulation in human oranimal patients.

It is further an object of the present invention to provide methods ofutilizing fibrinogen-binding proteins from S. epidermidis in order toreduce or prevent thrombin-induced coagulation and to enhance thedissolution of blood clots in human or animal patients.

It is another object of the invention to provide therapeuticcompositions based on fibrinogen-binding proteins from S. epidermidiswhich bind to the Bβ chain of fibrinogen which are useful in preventingor treating thrombin-induced coagulation in human or animal patients inneed thereof.

It is still further an object of the present invention to developcompositions from fibrinogen-binding proteins from S. epidermidis whichbind to the Bβ chain of fibrinogen, and which can block the thrombinbinding site on fibrinogen so as to be useful in methods of preventingcleavage of fibrinogen by thrombin and inhibiting the release offibrinopeptide B from fibrinogen.

These and other objects are provided by virtue of the present inventionwhich comprises compositions and methods which utilize the SdrG proteinfrom S. epidermidis, and other proteins which bind to the Bβ chain offibrinogen such as the A region of SdrG and the Fbe protein, in order totreat or prevent thrombin-induced coagulation in human or animalpatients. In addition, the invention comprises methods of administeringSdrG so as to treat or prevent a wide variety of conditions whereinblood coagulation can be dangerous or even life-threatening coagulation,including venous thrombosis, myocardial infarction and sickle cellcrisis episodes. The invention utilizes the ability of SdrG to inhibitthrombin-induced fibrin clot formation by inhibiting thrombin binding tofibrinogen and interfering with the release of fibrinopeptide B, andtherapeutic compositions containing an effective amount of SdrG can thusbe used as effective anti-coagulation agents. The SdrG compounds andcompositions of the present invention may also be used to reduce theconcentration of plasma fibrinogen in a patient's blood when so desired.

These embodiments and other alternatives and modifications within thespirit and scope of the disclosed invention are described in, or willbecome readily apparent from, reference to the detailed description ofthe preferred embodiments provided herein below.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a representation of the structural organization of SdrGwherein FIG. 1A is a representation of SdrG. The number of amino acidresidues contained in each region is indicated below each segment. S,signal sequence, A, N-terminal Fg binding region, B1 & B2, repeats ofunknown function, R, serine-aspartate repeat region, W, wall-spanningregion, M, membrane-spanning region. The positively-charged tail andLPXTG motif involved in cell-wall anchoring are also indicated; FIG. 1Bis a model of the recombinant His-tag construct rSdrG(50-597),representing the A region; and FIG. 1C shows a Coomasie stained SDS-PAGEof purified rSdrG(50-597).

FIG. 2 is a graphic representation of tests showing rSdrG(50-597)binding to immobilized Fg. Increasing concentrations of rSdrG(50-597)(●) were incubated with immobilized Fg in an ELISA. After incubation inthe wells for 1 h at room temperature, bound protein was detected asdescribed in the materials and method section. The apparent K_(D) was0.9×10⁻⁷ M. Values represent the mean±standard deviation of triplicatewells.

FIG. 3 is a graphic representation of tests showing localization of therSdrG(50-597) binding site in Fg using recombinant Bβ chain constructsin accordance with the invention. In FIG. 3A, models of the recombinanttruncates of the Fg Bβ chain constructed using the pQE30 His-tag vectoror the GST fusion vector pGEX-KG are shown. In FIG. 3B, whole E. colicell lysates containing the recombinant proteins were loaded onto a 10%SDS-polyacrylamide gel. The gel was transferred to a nitrocellulosemembrane and the blot was probed with biotin labeled rSdrG(50-597) anddeveloped as described in the materials and methods section. Lane 1,native Fg, lane 2, rβ(1-462), lane 3, rβ(1-341), lane 4, rβ(1-220), lane5, rβ(1-195), lane 6, rβ(25-195), lane 7, rβ(1-95), lane 8, rβ(25-95).

FIG. 4 shows results of tests indicating inhibition of rSdrG(50-597)binding to immobilized Fg by synthetic peptides in accordance with thepresent invention. In these tests, rSdrG(50-597) (50 nM) waspre-incubated with increasing concentrations of peptides for 1 h at roomtemperature and transferred to microtiter wells coated with 1 μg humanFg. After incubation in the wells for 1 h at room temperature, boundSdrG was detected as described in the materials and methods section. ForFIG. 4A: β1-25 (●), β6-25 (Δ), β1-25S (♦); FIG. 4B: β6-20 (▪), β1-20 (●)and β11-20 (◯). For FIG. 4C: FpA (□), FpB (σ), β-25 (●), β1-25S (♦).Values represent the mean±standard deviation of triplicate wells.

FIG. 5 shows rSdrG(50-597) binding to thrombin digested Fg. Fg coatedmicrotiter wells were pretreated for 30 min at 37° C. with thrombin (σ),thrombin and hirudin (●), hirudin alone (♦) or untreated (▪). Plateswere blocked, washed and incubated with biotin labeled rSdrG(50-597)(25-1000 nM) for 1 h at room temperature. Bound SdrG was detected asdescribed in materials and methods. Values represent the mean±standarddeviation of triplicate wells.

FIG. 6 is a quantitative analysis of rSdrG(50-597) binding to intactimmobilized Fg or Fg peptide β1-25. FIG. 6A shows increasingconcentrations of rSdrG(50-597) were incubated with thefluorescein-labeled N-terminal Bβ chain peptide β1-25 (10 nM) for 3 h inthe dark at room temperature. Equation 1 was used to fit the bindingdata. From three experiments the K_(D) for the interaction ofrSdrG(50-597) with peptide β1-25 was calculated to be 1.4±0.01×10⁻⁷ M.FIG. 6B: Binding of the fluorescein-labeled β1-25 to rSdrG(50-597) inthe presence of increasing concentrations of unlabeled β1-25 (●) or thescrambled Bβ chain peptide β1-25S (σ). Values are the mean of duplicatereactions.

FIG. 7 shows the inhibition of fibrin clot formation by rSdrG(50-597).Thrombin (1.0 NIH unit/ml) was added to a mixture of Fg (3.0 μM) andrSdrG(50-597) (●) (0-6.0 μM) or BSA (σ) (0-6.0 μM) in microtiter wells.Fibrin clot formation was monitored by measuring an increase in opticaldensity at 405 nm. Values represent the mean±standard deviation ofquadruple wells.

FIG. 8 shows the Inhibition of FpB release by rSdrG(50-597).Superimposed chromatograms show the amount of fibrinopeptide releasedwhen the Fg-thrombin sample has no SdrG present (upper curve) and whenthe Fg-thrombin sample is incubated with 1.5 μM rSdrG(50-597) (lowercurve) at the 60 min time point. The decrease in the amount of FpBreleased with SdrG present is shown in the lower curve.

FIG. 9 shows rSdrG(50-597) binding to Serine protease-digested Fg. Fgcoated microtiter wells were pretreated for 30 min at room temperaturewith ancrod (●), PBS (untreated) (σ), thrombin (♦) or contortrixobin(▪). Plates were blocked, washed and incubated with a serine proteaseinhibitor (1 NIH unit/ml hirudin for thrombin and 100 μg/ml PMSF forancrod and contortrixobin). Biotin labeled rSdrG(50-597) (25-1000 nM)was incubated in the wells for 1 h at room temperature. Values representthe mean±standard deviation of triplicate wells.

FIG. 10 shows the inhibition of FpB Release by rSdrG(50-597).Superimposed chromatograms show the amount of fibrinopeptide releasedwhen the Fg-contortrixobin sample has no SdrG present (upper curve) andwhen the Fg-contortrixobin sample is incubated with 1.5 μM rSdrG(50-597)(lower curve) at the 60 min time point. The decrease in the amount ofFpB released with SdrG present is shown in the lower curve.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there is provided a method andcomposition for treating or preventing thrombin-induced coagulationwherein an effective amount of SdrG which can bind the Bβ chain offibrinogen, or its active fragments such as the A region of SdrG, isadministered so as to inhibit the binding of thrombin to fibrinogen andreduce or prevent the release of fibrinopeptide B from fibrinogen andthus prevent the thrombin-induced clot formation process. In accordancewith the present invention, the inventors have now localized thefibrinogen-binding site for SdrG to the Bβ chain of the N-terminalregion of Fg, and more particularly to the region of from about residues6-20 on the Fg Bβ chain which is proximal to the thrombin cleavage site.Accordingly, the present invention provides for methods of using SdrG asan anti-coagulation agent since it inhibits thrombin binding, thuspreventing the cleavage and subsequent release of fibrinopeptide B whichis an initial step in the initiation of the production of fibrin and thethrombin-induced formation of blood clots.

SdrG is a fibrinogen-binding protein from S. epidermidis which has abinding region known as the A region or A domain at residues 50-597 ofthe SdrG protein. Detailed information concerning SdrG and its primarybinding region, known as the A domain or A region, has been disclosed inpending U.S. Ser. No. 09/386,962, filed Aug. 31, 1999, incorporatedherein by reference. The SdrG protein suitable for use in the presentinvention, which includes binding regions of the SdrG protein includingthe A domain or region, may be prepared through isolation of the naturalprotein or binding region, or more preferably through recombinant meansusing nucleic acids coding for SdrG and/or its binding region A. Thenucleic acid and amino acid sequences for SdrG and its binding region Ahave been previously disclosed in pending U.S. Ser. No. 09/386,962,filed Aug. 31, 1999 as discussed above, and these sequences may beutilized in conventional recombinant procedures in order to produce SdrGand/or its binding region A which will be suitable for use in thepresent invention.

In accordance with one specific embodiment of the present invention, arecombinant SdrG A region was obtained using plasmid cloning of an sdrGgene fragment. In this procedure, Escherichia coli strain JM101 was usedfor plasmid cloning. E. coli strain Topp3 (Stratagene) was used forprotein expression. Strains harboring plasmids were grown in Lennox Lbroth (Sigma) or on Lennox L agar (Sigma) supplemented with 100 μg/mlampicillin. The gene fragment encoding the entire A-region was amplifiedby PCR using S. epidermidis K28 genomic DNA as a template. Theoligonucleotide primers used were 5′-CCCGGATCCGAGGAGAATACA GTACAAGACG-3′(SEQ ID NO:2) and 5′-CCCGGTACCGATTTTTTCAGGAGGCAAGTCACC-3′ (SEQ ID NO:3).The restriction enzyme cleavage sites (underlined) BamHI and KpnI wereincorporated into the forward and reverse primers, respectively. Thereactions were carried out using a Perkin-Elmer DNA thermocyclcer. Thereactions contained 50 ng of template DNA, 100 pmol of forward andreverse primers, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, 10 mM KCl, 10 mM(NH₄)₂SO₄, 0.1% Triton X-100, 25 mM of each dNTP, and 5 units of Pfu DNApolymerase (Stratagene). Amplification was performed at 94° C. for 1min, 50° C. for 1 min, 72° C. for 4 min, for 25 cycles.

Next, the cloning of sdrG into the expression plasmid was carried out.In this process, the amplified sdrG fragment was digested with BamHI andKpnI and ligated into the expression plasmid pQE30 (Qiagen Inc.) thathad been digested with the same enzymes, yielding the constructpSdrG(50-597). The recombinant protein rSdrG(50-597) expressed from thisplasmid contains an N-terminal extension of six histidine residues(His-tag). Expression and purification of the recombinant protein wasthen obtained. In these steps, E. coli transformed with pSdrG(50-597)was grown for ˜2 h for the cultures to give an OD₆₀₀ of 0.6.rSdrG(50-597) expression was induced by the addition ofisopropyl-β-thiogalactopyranoside (IPTG) (Gibco-BRL) (225 μM) and thecultures were incubated at 37° C. for an additional 3 h. Bacteria werepelleted and resuspended in phosphate buffered saline (PBS), pH 7.5 (140mM NaCl, 270 μM KCl, 430 μM Na₂HPO₄, 147 μM KH₂PO₄) and frozen o/n at−20° C.

Next, bacterial cells were thawed and mechanically lysed by using aFrench Pressure Cell (SLM Amnico). Cell debris was removed bycentrifugation and filtration through a 0.45 μm filter membrane. Thesupernatant containing the recombinant protein was applied to a Ni²⁺charged (87.5 mM) 5 ml Hi Trap chelating column (Amersham PharmaciaBiotech) connected to a FPLC system. The column was equilibrated withbuffer A (0.1 M NaCl, 10 mM Tris-HCl, pH 8.0) before the application ofthe filtered lysate. The column was then washed with 10 bed volumes ofbuffer A containing 5 mM imidazole. Bound protein was eluted with acontinuous linear gradient of imidazole (5-120 mM; total volume 160 mls)in buffer A. Fractions were monitored for protein by determining theabsorbance at 280 nm and fractions containing rSdrG(50-597) wereidentified by SDS-PAGE (16). These fractions were pooled and dialyzedagainst PBS, pH 7.5. The dialyzed protein was then applied to aQ-Sepharose column (Amersham Pharmacia Biotech) equilibrated with 25 mMTris-HCl, pH 8.0. Bound protein was eluted with a continuous lineargradient of NaCl (0-0.5 mM; total volume 160 mls) in 25 mM Tris-HCl, pH8.0. Fractions containing the purified rSdrG(50-597) were identified bydetermining the absorbance at 280 nm and by SDS-PAGE. The truncated Aregion of ClfA was purified as previously reported (17).

In accordance with the present invention, the SdrG proteins or SdrG Aregion from S. epidermidis may be utilized as compositions to treat orprevent thrombin-induced coagulation in human or animal patients, andthus to treat or prevent a wide variety of conditions associatedtherewith. In the preferred composition, the SdrG protein is utilized inan amount effective to treat or prevent thrombin-induced coagulation,and the composition comprises the effective amount of the SdrG proteinalong with a pharmaceutically acceptable vehicle, carrier or excipientas would be well known to those of ordinary skill in the art includingsuch materials as saline, dextrose, water, glycerol, ethanol, othertherapeutic compounds commonly used, and combinations thereof. As oneskilled in this art would recognize, the particular vehicle, excipientor carrier used will vary depending on the nature of the patient and thepatient's condition, and a variety of modes of administration would besuitable for the compositions of the invention, as would be recognizedby one of ordinary skill in this art. Suitable methods of administrationof any pharmaceutical composition disclosed in this application willpreferably be intravenous, but other suitable methods of administering acompound for the desired purpose or preventing or reducingthrombin-induced coagulation may be introduced in other suitable ways aswould be known to those of ordinary skill in this art.

As indicated above, it is preferred that the compositions and methods inaccordance with the invention comprise SdrG or its A region in an amounteffective to prevent or reduce thrombin-induced coagulation in the bloodand thus be effective in the treatment or prevention of thrombin-inducedcoagulation under conditions such as venous thrombosis wherein suchtreatment or prevention is highly desirable. By effective amount ismeant that level of use of the SdrG proteins of the present inventionthat will be sufficient to prevent or reduce thrombin-inducedcoagulation in accordance with the invention, and thus be useful in thetreatment or prevention of a condition wherein thrombin-inducedcoagulation is sought to be prevented or alleviated. As would berecognized by one of ordinary skill in this art, the particular amountof the SdrG protein to be used in accordance with the invention to treator prevent thrombin-induced coagulation or a condition characterizedthereby will vary depending on the nature and condition of the patient,and/or the severity of the pre-existing condition to be treated orprevented, such as in an operation wherein blood coagulation isdisfavored.

In accordance with the present invention, a method is thus provided fortreating or preventing thrombin-induced coagulation of blood comprisingadministering to a human or animal patient in need thereof an SdrGprotein such as the binding region A of SdrG in an amount effective toprevent or reduce thrombin-induced coagulation in the blood. The SdrGprotein may be used in the form of a therapeutic,pharmaceutically-acceptable composition as described above, the amountutilized will be the amount effective in treating or preventingthrombin-induced coagulation as also described in more detail above. Asalso indicated above, the SdrG protein utilized in the invention ispreferably a recombinant protein, and in the particularly preferredembodiment, a recombinant SdrG protein is used which constitutes theSdrG A region and which has the sequence of the residues 50-597 of SdrG.In the preferred method, the SdrG compounds and compositions of theinvention are administered in any suitable way to effect introduction ofthe active agent into the patient's bloodstream or other applicable areain order to achieve the desired goal of reducing or preventingthrombin-induced coagulation in a human or animal patient. As one ofordinary skill in this art would recognize, this can be accomplished ina number of suitable ways, including direct intravenous or intraarterialinjection, or via injection into other target areas where theanti-coagulant effects of the compositions of the invention are needed.For example, the present compositions may be utilized in the same mannerthat heparin is introduced into a patient to achieve anti-coagulationeffects, e.g., through an intravenous injection or in other suitableways. In the preferred method, the SdrG compositions are administeredfor as long as necessary to achieve the desired anti-coagulant effect aswould be determined, e.g., by the physician or other health careprofessional administering such treatment to a patient. This could beaccomplished both in the treatment of a condition wherein therapeuticanti-coagulant treatment is necessary or where preventive treatment isneeded such as in an operation wherein maximization of anti-coagulativeeffects is desired.

Accordingly, the present invention contemplates administration ofeffective amounts of the fibrinogen binding compositions of theinvention as necessary to achieve a result associated with theinhibition of thrombin-induced coagulation, such as the prevention orreduction in binding of thrombin to fibrinogen, the interference orinhibition of the release of fibrinopeptide B from fibrinogen, or thetreatment or prevention of coagulation during a disease condition suchas venous thrombosis, myocardial infarction, etc. In accordance with thepresent invention, it is contemplated that fibrinogen-binding proteinsfrom S. epidermidis may be used to prevent or treat thrombin-inducedcoagulation wherein said fibrinogen-binding proteins, such as SdrG orFbe, or their respective A domains, are capable of binding the Bβ chainof fibrinogen. More particularly, the invention contemplates thatfibrinogen-binding proteins, or their active subregions such as the Adomains from SdrG or Fbe, which can bind at the site from about residues6 to 20 on the Bβ chain of fibrinogen will be useful in methods toprevent or treat thrombin-induced coagulation and the conditionsassociated therewith. In these methods, an effective amount of thefibrinogen-binding protein is preferably administered in order toachieve the desired result of reducing or preventing thrombin-inducedcoagulation, and said fibrinogen-binding proteins may be utilized incompositions containing an effective amount of the active agent alongwith a pharmaceutical acceptable vehicle, carrier or excipient.

The examples which follow are provided which relate to certain aspectsof the present invention and which exemplify certain aspects of thepresent invention. However, it will be appreciated by those of skill inthe art that the techniques disclosed in the examples are only exemplaryof techniques associated with the present invention, and that those ofordinary skill in the art recognize that, in light of the teachings ofthe present specification, many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

EXAMPLES Example 1 Preparation of Recombinant SdrG and the Determinationof its Binding Attributes in Accordance With the Present InventionSUMMARY

Staphylococcus epidermidis is an important opportunistic pathogen and isa major cause of foreign body infections. We have characterized theligand-binding activity of SdrG, a fibrinogen-binding MSCRAMM from S.epidermidis. Western ligand blot analysis showed that a recombinant formof the N-terminal A-region of SdrG bound to the native Bβ chain offibrinogen (Fg) and to a recombinant form of the Bβ chain expressed inE. coli. By analyzing recombinant truncates and synthetic peptidemimetics of the Fg Bβ chain, the binding site for SdrG was localized toresidues 6-20 of this polypeptide. Recombinant SdrG bound to a synthetic25 amino acid peptide (β1-25) representing the N-terminus of the Fg Bβchain with a K_(D) of 1.4×10⁻⁷ M as determined by fluorescencepolarization experiments. This was similar to the apparent K_(D)(0.9×10⁻⁷ M) calculated from an ELISA where SdrG bound immobilized Fg ina concentration dependent manner. SdrG could recognize fibrinopeptide B(residues 1-14), but with a substantially lower affinity than thatobserved for SdrG binding to synthetic peptides β1-25 and β6-20.However, SdrG does not bind to thrombin digested Fg. Thus, SdrG appearsto target the thrombin cleavage site in the Fg Bβ chain. In fact, SdrGwas found to inhibit thrombin-induced fibrinogen coagulation byinterfering with fibrinopeptide B release.

INTRODUCTION

Coagulase-negative staphylococci (CNS) are important opportunisticpathogens that are particularly associated with foreign body infectionsin humans. Staphylococcus epidermidis is the most common pathogenicspecies of CNS¹ and accounts for 74-92% of the infections caused by thisgroup of staphylococci (1).

FOOTNOTES¹The abbreviations used are: CNS, coagulase-negative staphylococci, ECM,extracellular matrix, Fg, fibrinogen, MSCRAMM, microbial surfacecomponent recognizing adhesive matrix molecules, ClfA and ClfB, clumpingfactors A and B, FnbpA and FnbpB, fibronectin-binding proteins A and B,SdrF and SdrG, serine-aspartate repeat proteins F and G, FpA and FpB,fibrinopeptides A and B, ELISA, enzyme-linked immunosorbent assay, HPLC,high performance liquid chromatography, PCR, polymerase chain reaction,PAGE, polyacrylamide gel electrophoresis, K_(D), equilibriumdissociation constant.

The molecular pathogenesis of most infections is complex and involvesmultiple microbial factors and host components, but is generallyinitiated by the adherence of the microbe to host tissues. Bacterialadherence involves specific surface components called adhesins.Bacterial pathogens, such as staphylococci that live in theextracellular space of the host, target extracellular matrix (ECM)components, including fibrinogen (Fg) and fibronectin, for adherence andcolonization. This process is mediated by a sub-family of adhesins thathave been termed MSCRAMMs (microbial surface components recognizingadhesive matrix molecules) (2). Staphylococcus aureus expresses multipleMSCRAMMs of which several have been characterized in some detail (For arecent review see Ref. 3).

In addition to S. epidermidis, S. aureus also causes serious foreignbody infections. S. aureus appears to adhere to the biomaterial throughan indirect mechanism. Upon implantation, the foreign body rapidlybecomes coated with host proteins derived primarily from plasma with Fgbeing a dominant component. S. aureus appears to adhere to the absorbedproteins rather than to the biomaterial itself using adhesins of theMSCRAMM family (4,5). At least four of the S. aureus MSCRAMMs recognizeFg. Two of these MSCRAMMs, clumping factor A and B (ClfA, ClfB), haveFg-binding A-regions followed by a long segment of Ser-Asp (SD)dipeptide repeats. The other two Fg-binding MSCRAMMs, contain a similarligand binding A-region followed by a fibronectin binding motif that isrepeated 5 times (6). Because the fibronectin binding activity wasidentified first, these two MSCRAMMs are known as fibronectin bindingprotein A and B (FnbpA and FnbpB) (7,8). Studies have demonstrated theimportance of ClfA and ClfB in the adherence of S. aureus toplasma-coated biomaterials. S. aureus mutants deficient in one or bothof these MSCRAMMs exhibited an impaired ability to adhere toplasma-coated catheters in vivo or ex vivo (9,10).

For S. epidermidis, adherence to foreign bodies could involve bothspecific and non-specific processes. The bacteria may initiallyassociate directly with the foreign body through non-specificinteractions, while the later stages of adherence may involve morespecific interactions between bacterial adhesins and host ligands. S.epidermidis expresses polysaccharide adhesins including PS/A and PIA,which are encoded by the ica locus (11,12). In addition, we (13) andothers (14) have recently shown that S. epidermidis contains surfaceproteins structurally related to S. aureus MSCRAMMs. Two of these S.epidermidis proteins, called SdrF and SdrG, have features typical ofGram-positive bacterial proteins that are anchored to the cell wall.Both proteins show significant amino acid sequence homology to ClfA andClfB from S. aureus including an ˜500 amino acid long A region, a SDdipeptide repeat region and features required for cell wall anchoring,including a LPXTG motif (FIG. 1A). Recent studies by Pei, et al. suggestthat an S. epidermidis protein called Fbe can bind Fg and specificallyrecognizes the Bβ chain of this molecule (15). In the current study, wehave localized the SdrG binding site in the Fg Bβ chain to theN-terminal segment of this polypeptide, proximal to the thrombincleavage site. In fact, we have demonstrated that SdrG inhibitsthrombin-induced fibrin clot formation by interfering with the releaseof fibrinopeptide B.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions-Escherichia Coli strain JM101was used for plasmid cloning. E. coli strain Topp3 (Stratagene) was usedfor protein expression. Strains harboring plasmids were grown in LennoxL broth (Sigma) or on Lennox L agar (Sigma) supplemented with 100 μg/mlampicillin.

PCR Amplification of the sdrG Gene Fragment—The gene fragment encodingthe entire A-region was amplified by PCR using S. epidermidis K28genomic DNA as a template. The oligonucleotide primers used were5′-CCCGGATCCGAGGAGAATACA GTACAAGACG-3′ (SEQ ID NO:2) and5′-CCCGGTACCGATTTTTTCAGGAGGCMGTCACC-3′ (SEQ ID NO:3). The restrictionenzyme cleavage sites (underlined) BamHI and KpnI were incorporated intothe forward and reverse primers, respectively. The reactions werecarried out using a Perkin-Elmer DNA thermocyclcer. The reactionscontained 50 ng of template DNA, 100 pmol of forward and reverseprimers, 20 mM Tris-HCl (pH 8.8), 2 mM MgSO₄, 10 mM KCl, 10 mM(NH₄)₂SO₄, 0.1% Triton X-100, 25 mM of each dNTP, and 5 units of Pfu DNApolymerase (Stratagene). Amplification was performed at 94° C. for 1min, 50° C. for 1 min, 72° C. for 4 min, for 25 cycles.

Cloning of sdrG into the Expression Plasmid—The amplified sdrG fragmentwas digested with BamHI and KpnI and ligated into the expression plasmidpQE30 (Qiagen Inc.) that had been digested with the same enzymes,yielding the construct pSdrG(50-597). The recombinant proteinrSdrG(50-597) expressed from this plasmid contains an N-terminalextension of six histidine residues (His-tag).

Expression and Purification of Recombinant MSCRAMM Protein-E. colitransformed with pSdrG(50-597) was grown for ˜2 h for the cultures togive an OD₆₀₀ of 0.6. rSdrG(50-597) expression was induced by theaddition of isopropyl-β-thiogalactopyranoside (IPTG) (Gibco-BRL) (225μM) and the cultures were incubated at 37° C. for an additional 3 h.Bacteria were pelleted and resuspended in phosphate buffered saline(PBS), pH 7.5 (140 mM NaCl, 270 μM KCl, 430 μM Na₂HPO₄, 147 μM KH₂PO₄)and frozen o/n at −20° C. Bacterial cells were thawed and mechanicallylysed by using a French Pressure Cell (SLM Amnico). Cell debris wasremoved by centrifugation and filtration through a 0.45 μm filtermembrane. The supernatant containing the recombinant protein was appliedto a Ni²⁺ charged (87.5 mM) 5 ml Hi Trap chelating column (AmershamPharmacia Biotech) connected to a FPLC system. The column wasequilibrated with buffer A (0.1 M NaCl, 10 mM Tris-HCl, pH 8.0) beforethe application of the filtered lysate. The column was then washed with10 bed volumes of buffer A containing 5 mM imidazole. Bound protein waseluted with a continuous linear gradient of imidazole (5-120 mM; totalvolume 160 mls) in buffer A. Fractions were monitored for protein bydetermining the absorbance at 280 nm and fractions containingrSdrG(50-597) were identified by SDS-PAGE (16). These fractions werepooled and dialyzed against PBS, pH 7.5. The dialyzed protein was thenapplied to a Q-Sepharose column (Amersham Pharmacia Biotech)equilibrated with 25 mM Tris-HCl, pH 8.0. Bound protein was eluted witha continuous linear gradient of NaCl (0-0.5 mM; total volume 160 mls) in25 mM Tris-HCl, pH 8.0. Fractions containing the purified rSdrG(50-597)were identified by determining the absorbance at 280 nm and by SDS-PAGE.The truncated A region of ClfA was purified as previously reported (17).

Synthetic Peptides—The synthetic Fg peptides β1-25, β1-25S, β1-20,β6-25, were custom ordered from Research Genetics and thefibrinopeptides A and B (FpA and FpB) were from Bachem. Peptides β6-20and β11-20 were synthesized in our laboratory using a multiple peptidesynthesizer by Advanced Chemtech. For the following peptides the residuenumbers are given and the sequence follows (Residue 1 corresponds to thefirst residue of the mature Bβ chain.): peptide β1-25, is composed ofthe first 25 amino acid residues of the N-terminus of the Bβ chain of Fg(QGVNDNEEGFFSARGHRPLDKK REE) (SEQ ID NO:4), peptide 1-20(QGVNDNEEGFFSARGHRPLD) (SEQ ID NO:5), peptide β6-25(NEEGFFSARGHRPLDKKREE) (SEQ ID NO:6), peptide β1-25S is a scrambledversion of peptide β1-25 (FSERKDLHQGEGNPREFVENDAKGR) (SEQ ID NO:7),peptide β6-20 (NEEGFFSA RGHRPLD) (SEQ ID NO:8), peptide β11-20(FSARGHRPLD) (SEQ ID NO:9), FpA (ADSEGEGDFLAEGGGVR) (SEQ ID NO:10), andFpB (QGVNDNEEGFFSAR) (SEQ ID NO:11). Peptides were purified by HPLC andanalyzed by MALDI mass spectrometry.

ELISA—Microtiter plates (Immulon 4, Dynatech Laboratories Inc.) werecoated with 1 μg of Fg (Enzyme Research Labs) in PBS, pH 7.5 for 18 h at4° C. Plates were washed three times with PBS, 0.05% Tween 20 (PBST) andblocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at roomtemperature. Plates were washed three times with PBST and rSdrG(50-597),diluted into PBS, was added to the wells and the plate was incubated for1 h at room temperature. Plates were washed three times with PBST andbound rSdrG(50-597) was detected by adding a 1:2000 dilution of ananti-His-tag mAb (Clontech) in PBST, 0.1% BSA. Plates were incubated for1 h at room temperature and then washed three times with PBST. A 1:2000dilution of goat anti-mouse alkaline phosphatase (AP)-conjugatedpolyclonal antibodies (Bio-Rad) in PBST, 0.1% BSA were added to thewells and the plate was incubated for 1 h at room temperature. Plateswere washed three times with PBST and developed with p-nitrophenylphosphate (Sigma) in 1 M diethanolamine, 0.5 mM MgCl₂, pH 9.0 at roomtemperature for ˜30 min. Plates were read at 405 nm using an ELISA platereader (Thermomax microplate reader, Molecular Devices).

In the inhibition experiments, 50 nM rSdrG(50-597) in PBS waspre-incubated with the indicated amounts of selected peptides for 1 h atroom temperature. The sample mixtures were added to the Fg-coated wellsand bound rSdrG(50-597) was detected as described above.

For the ELISA with thrombin-digested Fg, the plate was coated with Fgand blocked as described above. The plate was washed three times withPBST and 50 μl of 1.0 NIH unit/ml of thrombin was added to the Fg coatedwells. The plate was incubated at 37° C. for 30 min. The plate waswashed three times with PBST and 50 μl of 1.0 NIH unit/ml of hirudin(Sigma) was added to the wells and incubated at 37° C. for 30 min. Theplate was washed three times with PBST and blocked with 1% BSA for 1 hat room temperature. After washing three times with PBST, 100 μl ofbiotin labeled rSdrG(50-597) (25-1000 nM) or a rSdrG(50-597)/hirudin(1.0 NIH unit/ml) mixture was added to the wells and incubated for 1 hat room temperature. The plate was washed three times with PBST and a1:5000 dilution of streptavidin-AP conjugated (Boehringer Mannheim) inPBST/0.1% BSA was added to the wells for 1 h at room temperature. Theplate was washed three times with PBST and developed as described above.

Construction of Fg Bβ Chain Truncates—An E. coli strain harboringplasmid p668 which contains the cDNA for the Fg Bβ chain was kindlyprovided by Dr. Susan T. Lord (University of North Carolina, ChapelHill, N.C.). The 1525 bp fragment from p668 was subcloned into theplasmid pQE30 to produce recombinant mature Bβ chain with a N-terminalHis-tag. Additional Bβ chain constructs (FIG. 3A) were made bysubcloning into either pQE30 or pGEX-KG (Pharmacia) to producerecombinant proteins with a N-terminal His-tag or GlutathioneS-transferase (GST) fusion.

Western Ligand Blot Analysis—Whole E. coli lysates harboring eachrespective Fg Bβ chain construct were fractionated by SDS-PAGE and theseparated proteins were transferred to nitrocellulose membrane with asemi-dry transfer cell (Bio-Rad). The membrane was incubated overnightwith 5% (w/v) non-fat dry milk in PBS, pH 7.5 at 4° C. to saturatenon-specific binding sites. After blocking, the membrane was washedthree times with PBST and then incubated with biotin labeledrSdrG(50-597) (0.5 μM) for 1 h at room temperature. rSdrG(50-597) wasbiotin labeled using EZ Link-sulfo-NHS-LC biotin (Sigma) according tothe manufacturers' instructions. After three more washes with PBST, theblot was incubated with a 1:5000 dilution of streptavidin-AP conjugatedfor 1 h at room temperature and developed with5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium(NBT) (Biorad) in carbonate:bicarbonate buffer (14 mM Na₂CO₃, 36 mMNaHCO₃, 5 mM MgCl₂:6H₂O, pH 9.8) for 15 min at room temperature.

Fluorescence Polarization-Fluorescence polarization was used todetermine the equilibrium dissociation constant (K_(D)) for theinteraction of rSdrG(50-597) with peptide β1-25. The peptide was labeledwith fluorescein as previously described (18). Increasing concentrationsof rSdrG(50-597) in PBS, pH 7.5, were incubated with 10 nM labeledpeptide for 3 h in the dark at room temperature. Reactions were allowedto reach equilibrium. Polarization measurements were taken with aLuminescence Spectrometer LS50B (Perkin Elmer) using FL WinLab software(Perkin Elmer). Binding data was analyzed by nonlinear regression usedto fit a binding function as defined by the following equation:$\begin{matrix}{{\Delta\quad P} = {\frac{\Delta\quad{P_{\max} \cdot \lbrack{protein}\rbrack}}{K_{D} + \lbrack{protein}\rbrack}.}} & {{Equation}\quad 1}\end{matrix}$where ΔP corresponds to the change in fluorescence polarization,ΔP_(max) is the maximum change in fluorescence, and K_(D) is theequilibrium dissociation constant of the interaction. A single bindingsite was assumed in this analysis.

Fg Coagulation Assay—150 μl of a 3.0 μM Fg solution was incubated with10 μl of rSdrG(50-597) or BSA (1.0-6.0 μM) and 50 μl of thrombin (Sigma)(1.0 NIH unit/ml) in microtiter wells at room temperature. Clotformation was monitored by measuring the increase in optical density(OD) at 405 nm over time and expressed as V_(max) (mOD/min). A platereader (Thermomax microplate reader, SOFTmax software, MolecularDevices) was used to monitor OD. Using the kinetic mode with onewavelength (L1=405 nm), samples were read every 10 sec for 5 min. Inthis assay, 1.0 NIH unit/ml of thrombin incubated with 3.0 μM Fgproduced a fibrin clot in 5 min at room temperature.

Release of Fibrinopeptides by Thrombin—The thrombin catalyzed release offibrinopeptides A and B was analyzed as follows. Fg solutions werediluted to 0.3 μM in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mMε-aminocapriotic acid, and 1.0 mM CaCl₂. ε-aminocapriotic acid wasincluded to inhibit any possible plasmin contaminant activity. Thrombinwas added to a final concentration of 0.05 NIH units/ml. rSdrG(50-597)was added to a final concentration of 0.3 μM, 0.6 μM, or 1.5 μM. Thetubes were mixed by inversion and 500 μl aliquots were removed for the15 and 60 min time points. The aliquots were incubated at roomtemperature and then immersed in boiling water for 15 min to halt thereaction. The aliquots were stored on ice for the remainder of the timecourse. At the end of the reaction the samples were centrifuged for 15min at 4° C., and the supernatants were removed and stored at −20° C.overnight prior to analysis by high performance liquid chromatography.

The fibrinopeptides released were monitored by reverse phase HPLCessentially as described (19). The samples were loaded onto a WatersDelta-Pak C₁₈ column equilibrated with buffer A (25 mM NaH₂PO₄/Na₂HPO₄,pH 6.0). Fibrinopeptides were eluted with a linear gradient from 100%buffer A to 40% buffer B (Buffer A with 50% acetonitrile) and monitoredby absorbance at 205 nm. Fibrinopeptide peak area was determined usingthe software Waters Millennium³².

RESULTS

Expression and Purification of Recombinant SdrG A-region—In order tocharacterize the ligand binding activity of SdrG, a recombinant form ofthe putative ligand binding A-region (residues 50-597) (FIG. 1C) wasexpressed in E. coli with a N-terminal His-tag. This protein construct,rSdrG(50-597), was purified by metal chelate affinity chromatographyfollowed by ion-exchange chromatography. The purity of the recombinantprotein was confirmed by SDS-PAGE analysis, where it migrated with anapparent molecular mass of 97 kDa (FIG. 1B). This is larger than thetheoretical molecular weight of 63.7 kDa predicted from the primaryamino acid sequence of this protein. Analysis of rSdrG(50-597) by MALDImass spectrometry indicated a molecular mass of 63.3 kDa. Aberrantmigration in SDS-PAGE has also been observed with recombinant MSCRAMMsderived from S. aureus, and may be explained by the 15 hydrophilicnature of these proteins (9,13).

SdrG Binds the Fg Bβ Chain—SdrG is closely related to the recentlydescribed fibrinogen binding MSCRAMM Fbe (15). Therefore, we initiallyexamined the ligand binding specificity of SdrG for Fg in an ELISA. Inthis assay, rSdrG(50-597) bound immobilized Fg, but failed to bind toother immobilized ECM proteins such as fibronectin, collagen types I andIV, vitronectin, laminin and thrombospondin (data not shown). Binding ofincreasing concentrations of rSdrG(50-597) to absorbed Fg exhibitedsaturation kinetics (FIG. 2). Together these observations demonstratethe specificity of the SdrG-Fg interaction. Furthermore, biotin labeledrSdrG(50-597) recognized the Bβ chain, but not the Aα or γ chains of Fgwhen analyzed by Western ligand blotting (data not shown).

Localization of the SdrG Binding Site in the Fg Bβ Chain—The observationthat rSdrG binds the Fg Bβchain fractionated under reducing anddenaturing conditions in Western ligand blot analysis suggests that theMSCRAMM recognizes a specific linear amino acid sequence in the Bβchain. To explore this possibility and locate the SdrG binding site, arecombinant mature Fg Bβ chain and a series of truncated forms of the Bβchain expressed in E. coli were analyzed by Western ligand blot. Therecombinant Bβ chain constructs were expressed as either His-tag orglutathione S-transferase (GST) fusion proteins (FIG. 3A). Thefractionated proteins were transferred to a supporting membrane andprobed with biotin labeled rSdrG(50-597) (FIG. 3B). rSdrG(50-597)recognized the mature recombinant Bβ chain (residues 1-462) as well asthe recombinant truncates encompassing residues 1-341, 1-220, 1-195 and1-95.

However, rSdrG(50-597) failed to bind to the two recombinant truncatesthat lacked the N-terminal 25 amino acid residues of the Bβ chain,rβ(25-95) and rβ(25-195) (FIG. 3B). These observations demonstrate thatrSdrG(50-597) recognizes a linear sequence in Fg and suggests that thissite lies within the N-terminal region of the Bβ chain.

Inhibition of rSdrG(50-597) Binding to Fg by Synthetic Peptides—Tofurther define the rSdrG(50-597) binding site in the Fg Bβ chain, weused a peptide mimetic approach. A series of peptides representingsegments of the N-terminal region of the FgBβ chain were synthesized andtested for their ability to inhibit the binding of rSdrG(50-597) to Fgin an ELISA (FIG. 4). In FIG. 4A, peptides β1-25 and β6-25 were shown toinhibit the binding of rSdrG(50-597) to Fg in a concentration dependentmanner, whereas the scrambled version of β1-25, peptide β1-25S, did notinterfere with the binding of rSdrG(50-597) to Fg. Effective inhibitionof rSdrG(50-597) binding to Fg was also observed with peptide β6-20 and,to a somewhat lesser degree, with β11-20. Peptide β11-20 was essentiallyinactive in this assay (FIG. 4B).

The thrombin cleavage sites in Fg lie between residues 14 (Arg) and 15(Gly) in the Bβ chain and between 16 (Arg) and 17 (Gly) in the Aα chain.Upon cleavage of Fg by thrombin the fibrinopeptides, FpA and FpB, aresequentially released. The fibrinopeptides were examined as inhibitorsof rSdrG(50-597) binding to Fg in an ELISA. FpB inhibited the binding ofrSdrG(50-597) in a concentration dependent manner, but this peptide wasat least 10 fold less active than the synthetic peptide β1-25 (FIG. 4C).FpA was essentially inactive and behaved similar to the scrambledpeptide β1-25S. Taken together, this suggest that rSdrG(50-597)recognizes a linear amino acid sequence in the Bβ chain located withinresidues 6-20. This recognition site appears to overlap the thrombincleavage site in this polypeptide.

rSdrG Binding to Thrombin-Digested Fg—The rSdrG binding site seems tolie within close proximity to the thrombin cleavage site, therefore, weinvestigated if rSdrG(50-597) could bind to Fg in which the thrombincleavage site was abolished. Fg coated microtiter wells were pretreatedwith thrombin or thrombin plus hirudin (which inhibits thrombinactivity) in order to remove FpB and destroy the cleavage site. Theability of rSdrG(50-597) to bind to this thrombin digested Fg wassignificantly impaired (FIG. 5), suggesting that the thrombin cleavagesite residues Bβ 14 (Arg), 15 (Gly) and residues within FpB (1-14) areessential for rSdrG(50-597) to bind Fg.

Determination of Equilibrium Dissociation Constants (K_(D))—Anequilibrium dissociation constant (K_(D)) for the interaction ofrSdrG(50-597) with the Fg Bβ chain peptide β1-25 was determined. Byanalyzing the binding of increasing concentrations of rSdrG(50-597) tothe fluorescein-labeled β1-25 peptide in a fluorescence polarizationassay, rSdrG(50-597) binding to the labeled peptide exhibited saturationkinetics with a K_(D) of 1.4±0.01×10⁻⁷ M (FIG. 6A). To demonstrate thespecificity of this interaction, the binding of rSdrG(50-597) to thelabeled β1-25 peptide was measured in the presence of increasing amountsof unlabeled peptide (β1-25) or scrambled peptide (β1-25S). Theunlabeled β1-25 peptide, but not peptide β1-25S inhibited binding ofrSdrG(50-597) to the fluorescein-labeled β1-25 peptide, in aconcentration dependent manner (FIG. 6B). The apparent K_(D) determinedfor the binding of rSdrG(50-597) to the fluorescein labeled peptide31-25 is similar to the apparent K_(D) (0.9×10⁻⁷ M) for the interactionof rSdrG(50-597) with immobilized, intact Fg as determined by ELISA(FIG. 2).

rSdrG(50-597) Inhibits Thrombin-Induced Fibrin Clot Formation—In thefinal stages of the blood coagulation cascade, thrombin cleaves Fgreleasing the fibrinopeptides and producing fibrin monomers. Thesefibrin monomers then polymerize to form a fibrin clot (20). Thelocalization of the SdrG binding site described above raises thepossibility that rSdrG(50-597) may be able to inhibit thrombin-inducedfibrin clot formation, perhaps by directly competing with thrombin forbinding to the N-terminus of the Bβ chain of Fg or by binding to aproximal site and sterically blocking thrombin's proteolytic attack onthe Bβ chain. To test this hypothesis, we designed a fibrin clotinhibition assay in which 3.0 μM Fg, 0-6.0 μM rSdrG(50-597) and 1.0 NIHunit/ml of thrombin were incubated and the formation of a fibrin clotwas monitored by measuring the increase in optical density at 405 nm.FIG. 7 shows that rSdrG(50-597) inhibited fibrin clot formation in aconcentration dependent manner, whereas BSA had no effect. This suggeststhat rSdrG(50-597) can interfere with thrombin activity by binding to asite in the Fg Bβ chain that is proximal to or overlaps the binding sitefor thrombin.

Analysis of Fibrinopeptide B Release by HPLC—The release of FpA and FpBfrom the N-terminus of the Aα and Bβ chains of Fg by thrombin can bemonitored and quantitated by high performance liquid chromatography(19,21,22). We examined the effect of rSdrG(50-597) on fibrinopeptiderelease by measuring the peak areas of FpA and FpB, as detected by HPLC.The HPLC chromatograms shown in FIG. 8 show the expected fibrinopeptiderelease following digestion of Fg with thrombin superimposed with thefibrinopeptide release when Fg and thrombin are incubated withrSdrG(50-597). A significant decrease in the amount of FpB release wasshown with a 1:1 ratio of rSdrG(50-597) to Fg (Table I) whereas, a 5:1ratio was effectively able to inhibit the release of FpB (FIG. 8). Thiseffect was seen at an incubation time of 15 min and 60 min. There was noapparent interference of FpA release by rSdrG(50-597). TABLE IPercentage of FpB released in the presence of SdrG SdrG:Fg 15 min 60 min0:1 100 100 1:1 65.4 45.2 2:1 13.9 17.4 5:1 <0.001 <0.001Fg (0.3 μM) was incubated with SdrG and 0.5 NIH units/ml of thrombin atroom temperature and the samples were analyzed by HPLC. The amount offibrinopeptide released was determined by measuring the area under thepeaks on the HPLC chromatograms. The data was normalized in order tocompare the data from separate chromatograms assuming that the releaseof FpA is not affected by the presence of SdrG. The peak arearepresenting FpB in the absence of SdrG was set to 100%.

DISCUSSION

In this study, we have shown that SdrG binds the N-terminus of the Bβchain of Fg with a high degree of specificity. The binding of SdrG to anN-terminal Fg peptide exhibits a K_(D) of 1.4×10⁻⁷ M, which issignificantly lower than the K_(D) determined for the binding of ClfA toa γ chain peptide (2.0×10⁻⁵ M) (18). Thus, SdrG appears to have a higheraffinity for its respective synthetic Fg peptide target compared to theS. aureus MSCRAMM. The K_(D) determined for the binding of SdrG to thesynthetic peptide β1-25 is similar to the apparent K_(D) estimated forthe binding of SdrG to intact Fg absorbed onto microtiter wells. Thisobservation suggests that the SdrG binding site in the synthetic peptideis presented in a nearly optimal form and that additional segments of Fgdo not significantly contribute to the formation of the SdrG bindingsite.

Several studies have examined the role of Fg binding MSCRAMMs from S.aureus as virulence factors in animal models. Strains in which the genesencoding ClfA or ClfB have been inactivated are less virulent comparedto the wild type strain in a rat model of catheter-induced endocarditis(23,24). These results suggest that ClfA- and ClfB-mediated adherence isrequired for the maximum virulence potential of S. aureus to beexpressed. ClfB has been shown to promote S. aureus adherence to ex vivohemodialysis tubing, further confirming that ClfB contributes tobacterial attachment to biomaterials coated with host proteins (9). In arecent study, Stutzmann Meier, et al. showed that heterologousexpression of ClfA on Streptococcus gordonii, which is generallyconsidered a non-virulent bacterium, rendered this organism pathogenicin a rat endocarditis model (25). With the discovery that SdrG is a Fgbinding MSCRAMM expressed by S. epidermidis, the possibility arises thatSdrG can act as a virulence factor in S. epidermidis-induced infectionsand plays a role similar to that of the Fg binding MSCRAMMs in S.aureus-induced infections.

We have mapped the binding site of rSdrG(50-597) in the Fg Bβ chain to alinear sequence in the N-terminal region of this polypeptide. Peptideβ6-20 is a potent inhibitor of the binding of rSdrG(50-597) to Fg,whereas FpB (1-14) has poor inhibitory activity. Because peptide β6-20,but not β11-20 is recognized by this MSCRAMM, the N-terminal border ofthe binding site must lie between residues 6 and 11 of the Bβ chain. Theobservation that rSdrG(50-597) is unable to bind to thrombin digestedFg, i.e. the fibrinopeptides are absent, suggests that the C-terminus ofthis binding site is located between residues 14 and 20 of the Bβ chain.

It is striking that many of the identified staphylococcal MSCRAMMsappear to specifically recognize Fg, although the sites targeted in Fgby these proteins vary. ClfA, FnbpA and FnbpB of S. aureus all recognizethe C-terminus of the Fg γ chain (6,27). ClfB from S. aureus targets anas yet unidentified site in the Aα chain (9) and SdrG is here shown tobind to the N-terminus of the Bβ chain. Thus, these MSCRAMMs use aconserved A region to bind different sites in Fg. Furthermore, theMSCRAMMs appear to target sites in Fg that are important in themolecular physiology of this key component of hemostasis. The C-terminusof the γ chain is recognized by the platelet integrin α_(IIb)β₃ and ClfAis a potent inhibitor of Fg-induced platelet aggregation (26,27). Here,we show that the binding site in the Bβ chain for rSdrG(50-597) appearsto overlap the thrombin cleavage site and that rSdrG(50-597) caninterfere with fibrin clot formation by inhibiting the thrombin-inducedrelease of FpB. Fg may play an important role in the host's defenseagainst microbial infections and interfering with this function givesthe bacteria an advantage and the ability to survive in a hostileenvironment. One such potential advantage may be related to the observedchemotactic activity of FpB for human peripheral blood leukocytes(28-30). We have shown that rSdrG(50-597) can prevent the release ofFpB, thus one can speculate that the reason S. epidermidis possesses aprotein that can bind to this region of the Fg Bβ chain is to preventthe release of chemotactic elements. This may reduce the influx ofphagocytic neutrophils and help to ensure the survival of the bacteriain the host.

REFERENCES

The following articles are incorporated herein by reference:

-   1. Garrett, D. O., Jochimsen, E., Murfitt, K., Hill, B., McAllister,    S., Nelson, P., Spera, R. V., Sall, R. K., Tenover, F. C., Johnston,    J., Zimmer, B., and Jarvis, W. R. (1999) Infect Control Hosp    Epidemiol 20(3), 167-70.-   2. Patti, J. M., and Höök, M. (1994) Curr Opin Cell Biol 6(5), 752-8-   3. Foster, T. J., and Höök, M. (1998) Trends Microbiol 6(12), 484-8-   4. Galliani, S., Viot, M., Cremieux, A., and Van der    Auwera, P. (1994) J Lab Clin Med 123(5), 685-92.-   5. Vaudaux, P., Pittet, D., Haeberli, A., Huggler, E., Nydegger, U.    E., Lew, D. P., and Waldvogel, F. A. (1989) J Infect Dis 160(5),    865-75.-   6. Wann, E. R., Gurusiddappa, S., and Höök, M. (2000) J Biol Chem    275(18), 13863-71.-   7. Flock, J. I., Fröman, G., Jonsson, K., Guss, B., Signas, C.,    Nilsson, B., Raucci, G., Hook, M., Wadstrom, T., and    Lindberg, M. (1987) Embo J 6(8), 2351-7.-   8. Fröman, G., Switalski, L. M., Speziale, P., and Höök, M. (1987) J    Biol Chem 262(14), 6564-71-   9. Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Höök, M.,    and Foster, T. J. (1998) Mol Microbiol 30(2), 245-57-   10. Vaudaux, P. E., Francois, P., Proctor, R. A., McDevitt, D.,    Foster, T. J., Albrecht, R. M., Lew, D. P., Wabers, H., and    Cooper, S. L. (1995) Infect Immun 63(2), 585-90-   11. McKenney, D., Hubner, J., Muller, E., Wang, Y., Goldmann, D. A.,    and Pier, G. B. (1998) Infect Immun 66(10), 4711-20-   12. Cramton, S. E., Gerke, C., Schnell, N. F., Nichols, W. W., and    Götz, F. (1999) Infect Immun 67(10), 5427-33.-   13. McCrea, K. W., Hartford, O., Davis, S., Ni Eidhin, D., Lina, G.,    Speziale, P., Foster, T. J., and Höök, M. (2000) Microbiology 146(Pt    7), 1535-46.-   14. Nilsson, M., Frykberg, L., Flock, J. I., Pei, L., Lindberg, M.,    and Guss, B. (1998) Infect Immun 66(6), 2666-73-   15. Pei, L., Palma, M., Nilsson, M., Guss, B., and    Flock, J. I. (1999) Infect Immun 67(9), 4525-30-   16. Laemmli, U. K. (1970) Nature 227, 680-685-   17. McDevitt, D., Francois, P., Vaudaux, P., and    Foster, T. J. (1995) Mol Microbiol 16(5), 895-907.-   18. O'Connell, D. P., Nanavaty, T., McDevitt, D., Gurusiddappa, S.,    Höök, M., and Foster, T. J. (1998) J Biol Chem 273(12), 6821-9.-   19. Mullin, J. L., Gorkun, O. V., Binnie, C. G., and    Lord, S. T. (2000) J Biol Chem 275(33), 25239-46.-   20. Herrick, S., Blanc-Brude, O., Gray, A., and Laurent, G. (1999)    Int J Biochem Cell Biol 31(7), 741-6-   21. Ng, A. S., Lewis, S. D., and Shafer, J. A. (1993) Methods    Enzymol 222, 341-58-   22. Haverkate, F., Koopman, J., Kluft, C., D'Angelo, A., Cattaneo,    M., and Mannucci, P. M. (1986) Thromb Haemost 55(1), 131-5.-   23. Moreillon, P., Entenza, J. M., Francioli, P., McDevitt, D.,    Foster, T. J., Francois, P., and Vaudaux, P. (1995) Infect Immun    63(12), 4738-43-   24. Entenza, J. M., Foster, T. J., Ni Eidhin, D., Vaudaux, P.,    Francioli, P., and Moreillon, P. (2000) Infect Immun 68(9), 5443-6.-   25. Stutzmann Meier, P., Entenza, J. M., Vaudaux, P., Francioli, P.,    Glauser, M. P., and Moreillon, P. (2001) Infect Immun 69(2),    657-664.-   26. Farrell, D. H., Thiagarajan, P., Chung, D. W., and    Davie, E. W. (1992) Proc Natl Acad Sci USA 89(22), 10729-32.-   27. McDevitt, D., Nanavaty, T., House-Pompeo, K., Bell, E., Turner,    N., McIntire, L., Foster, T., and Höök, M. (1997) Eur J Biochem    247(1), 416-24-   28. Kay, A. B., Pepper, D. S., and McKenzie, R. (1974) Br J Haematol    27(4), 669-77.-   29. Richardson, D. L., Pepper, D. S., and Kay, A. B. (1976) Br J    Haematol 32(4), 507-13.-   30. Senior, R. M., Skogen, W. F., Griffin, G. L., and    Wilner, G. D. (1986) J Clin Invest 77(3), 1014-9.

Example 2 Experiments Showing SdrG Inhibits Serine Protease Digestion ofHuman Fibrinogen

The localization of the binding site for SdrG in Fg revealed that thethrombin cleavage site was in close proximity to the SdrG binding site.We were able to show that SdrG could not bind to thrombin-treated Fg butcould, in fact, inhibit thrombin-catalyzed fibrin clot formation. In arelated study, we analyzed SdrG for its ability to bind to immobilizedFg that had been treated with other serine proteases isolated from snakevenom and for its ability to inhibit clot formation in the presence ofthese same proteases. The fibrinolytic activities of snake venoms havebeen well documented and their activities are similar to thrombin,however, some may be specific for the Aα chain or the Bβ chain only. Inthis study we employed three proteases from snake venoms that havedifferent activities. Ancrod, an α-fibrinogenase isolated fromCalloselasma rhodostoma (Malayan Pit viper), releases only FpA and leadsto the formation of an unstable fibrin clot (Bell 1997). Contortrixobin,a β-fibrinogenase isolated from Agkistrodon contortrix contortrix(Southern Copperhead) preferentially releases FpB but does not form aclot effectively because FpA has not been released.

RESULTS

SdrG binding to serine protease-treated immobilized Fg—In an ELISAsimilar to the ELISA experiment described previously, we found thatrSdrG(50-597) could not bind to Fg treated with thrombin orcontortrixobin, the β-fibrinogenase but could bind to untreated Fg.rSdrG(50-597) could actually bind better to Fg treated with ancrod, theα-fibrinogenase, than to untreated Fg (FIG. 9). The A□ chain mayinterfere with rSdrG(50-597) binding to the Bβ chain and thisobservation may be due to the absence of FpA, thus making the N-terminusof the Bβ chain more easily accessible.

Analysis of Fibrinopeptide B Release by HPLC— As in chapter two, wemonitored the fibrinopeptide release in the presence of SdrG and theβ-fibrinogenase contortrixobin. The HPLC chromatograms shown in FIG. 10show the expected fibrinopeptide release following digestion of Fg withcontortrixobin superimposed with the fibrinopeptide release when Fg andcontortrixobin are incubated with rSdrG(50-597). A decrease in theamount of FpB release is seen when a 1:5 ratio of Fg to rSdrG (50-597)is used. The amount of FpB released with contortrixobin is significantlyless than the amount released with thrombin. This is most likely becauseFpA is still intact on the Fg molecule, thus contortrixobin is not aseffective at cleaving Fg due to steric hindrance from FpA.

DISCUSSION

The results of this study with serine proteases isolated from snakevenoms corroborate the results seen with SdrG and thrombin.rSdrG(50-597) was not able to bind to Fg that was treated with adifferent protease that preferentially cleaves the FpB from the Bβchain. This eliminates any concern that the effect seen withthrombin-treated Fg was in any way due to thrombin inhibitingrSdrG(50-597) from binding. Because rSdrG(50-597) was still able to bindto ancrod-treated Fg this also confirms that the FpA from the Aa chainwas not involved in SdrG binding to Fg.

rSdrG(50-597) was able to effectively inhibit FpB release in thepresence of the β-fibrinogenase contortrixobin supporting the evidencethat was shown previously and substantiating the mechanism by which SdrGis inhibiting clot formation is in fact by inhibiting FpB release. Wewere, however, unable to perform the clot inhibition experiment shown inchapter two due to the ineffective clot formation when only FpB isreleased, thus there was no accurate control.

Interestingly, ancrod has been used clinically as an antithromboticagent for a number of different disease conditions including stroke,myocardial infarction, sickle-cell crisis and venous thrombosis¹ (Forbes1993; Atkinson 1997) (Gilles, Reid et al. 1968), (Davies, Merrick et al.1972). This defibrinating enzyme cleaves FpA, but not FpB from Fg toform a clot that is very sensitive to endogenous fibrinolysis,additionally ancrod activates plasminogen further contributing tofibrinolysis (Pizzo, Schwartz et al. 1972; Carr 1975; Bell 1997). Theresult of administering ancrod is a significant reduction in plasma Fgconcentration within minutes and within hours the level of Fg ismarkedly depressed. Hypofibrinogenemia is sustained by administeringancrod daily. After termination of treatment, the plasma Fg risesgradually, returning to normal levels in days (Bell, Bolton et al.1968). The limited clinical experience indicates that defibrination isachieved with ancrod with reasonable safety, however, the elaboration ofneutralizing antibodies with repeated injections of ancrod leads toresistance (Pitney, Holt et al. 1969; Pitney and Regoeczi 1970),(Vinazzer 1973; Sapru, Moza et al. 1975).¹Definitions: venous thrombosis—the presence of a blood clot within avein, hypofibrinogenemia—abnormal deficiency of Fg in the blood,thrombocytopenia-persistent decrease in the number of blood platelets

SdrG inhibits clot formation by preventing the release of FpB. Althoughthe mechanism of thrombosis prevention by SdrG is different than that ofancrod, the ability of SdrG to inhibit clot formation could potentiallylead to its use as a novel anti-thrombotic agent. Certainly, much moreresearch would be needed to reliably assess its effectiveness and safetyrelative to heparin. Heparin is the standard treatment for thromboticdisorders and its mode of action is by increasing the effectiveness ofanti-thrombin III. A potentially important indication for ancrod andpossibly SdrG may be to avoid heparin-induced thrombocytopenia,resulting from heparin treatment. In addition, since SdrG binds to theBβ chain of Fg, it may be possible to also use it in conjunction withother agents, such as ancrod, which target the Aα chain of Fg in orderto further enhance the anti-coagulation when necessary. If used in thisfashion, in addition to a composition of SdrG used to reduce or preventthrombin-induced coagulation, ancrod in an amount effective to interfereor inhibit the release of fibrinopeptide A from fibrinogen may also beadministered along with the SdrG.

All of the references disclosed herein are incorporated by reference.

1. A therapeutic composition for inhibiting thrombin-induced coagulationof blood in a human or animal patient in need thereof comprising an SdrGserine-aspartate repeat protein in an amount effective to inhibitthrombin-induced coagulation, and a pharmaceutically acceptable vehicle,carrier or excipient.
 2. A method of inhibiting thrombin-inducedcoagulation comprising administering to a human or animal patient inneed thereof the composition of claim 1 in an amount effective toinhibit thrombin-induced coagulation.
 3. A method for treating a diseasecondition selected from the group consisting of stroke, myocardialinfarction, sickle-cell crisis and venous thrombosis comprisingadministering to a human or animal patient in need thereof an effectiveamount of the composition of claim
 1. 4. A method for reducing plasmafibrinogen concentration in blood comprising administering to a human oranimal an effective amount of the composition according to claim
 1. 5. Atherapeutic composition for inhibiting thrombin-induced coagulationcomprising a fibrinogen-binding protein from S. epidermidis which canbind to the Bβ chain of fibrinogen in an amount effective to inhibitthrombin-induced coagulation, and a pharmaceutically acceptable vehicle,carrier or excipient.
 6. The therapeutic composition according to claim5 wherein the fibrinogen-binding protein is selected from the groupconsisting of SdrG, Fbe and their respective A regions.
 7. Thetherapeutic composition according to claim 5 wherein the SdrG A regionhas the sequence of residues 50-597 of SdrG.
 8. The therapeuticcomposition according to claim 5 wherein the fibrinogen-binding proteinis a recombinant protein.
 9. The therapeutic composition according toclaim 5 wherein the fibrinogen-binding protein binds at the site ofresidues 6-20 of the fibrinogen Bβ chain
 10. A method of inhibitingthrombin-induced coagulation comprising administering to a human oranimal patient in need thereof the composition of claim 5 in an amounteffective to inhibit thrombin-induced coagulation.
 11. A method fortreating a disease condition selected from the group consisting ofstroke, myocardial infarction, sickle-cell crisis and venous thrombosiscomprising administering to a human or animal patient in need thereof aneffective amount of the composition of claim
 5. 12. A method forreducing plasma fibrinogen concentration in blood comprisingadministering to a human or animal an effective amount of thecomposition according to claim 5.